Bacteriorhodopsin (BR) is a protein found in the cell membrane of Halobacterium halobium, a bacterium found naturally in environments having extremely high salt concentrations. The BR molecule can be isolated from the bacteria in the form of two-dimensional, highly organized sheets called "purple membrane." These sheets are the thickness of the BR molecule (about 50 .ANG.) and have an average area of 1 .mu.m.sup.2.
Purple membrane sheets are in most cases crystalline, having a two-dimensional hexagonal close-packed structure. At each crystal lattice site there are three BR molecules present (a trimer). Each purple membrane sheet contains 10,000 to 100,000 BR molecules, depending on its overall size. Purple membrane also contains other molecules, known as lipids, which are important to its structure and stability. Lipids comprise about 15% of the total weight of the sheet.
The BR molecule itself consists of two parts: (1) the retinal (or chromophore) component; and (2) the opsin component, which surrounds the retinal component. The BR molecule, unlike many proteins, is exceedingly stable, both to light and to heat. This stability arises primarily from the two-dimensional crystallinity of the purple membrane and from the cage effect of the opsin on the light-sensitive retinal component. Such crystallinity lends stability to the trimeric structure of the protein, and in fact denaturation occurs only at high temperatures and in extreme chemical environments. For example, the protein is stable above 70.degree. C. in an aqueous environment, and can be subjected to pH ranging from 1.0 to 12.0. Furthermore, the retinal chromophore is unusually photostable, because it is held tightly within a pocket inside the protein. The stability of BR makes it uniquely suited toward many practical optical recording applications.
Purple membrane BR is often referred to as the "normal" form of BR since it is found under near-neutral pH and naturally occurring cation concentrations. When BR is exposed to light within its absorption spectrum, the absorption of a photon induces several successive changes to both the retinal and the opsin components, which translate into many different photochromic shifts. These photochromic shifts are strongly correlated with (1) the configuration of the retinal component (trans or cis), (2) the conformational changes of the opsin component (and thus the local charge environment near the retinal component), and (3) the deprotonation/reprotonation of the Schiff base and its counter-ions.
BR materials can be modified or engineered by making alterations in the amino acid sequence of the "native" BR molecule using site-specific mutagenesis. These modifications can change the photochromic effects dramatically. For example, a widely investigated alteration, the D96N strain, in which an asparagine amino acid has been substituted for aspartate, leads to a dramatically lengthened photochromic lifetime by impeding the reprotonation of the Schiff base. See Downie and Smithey, 21 Opt. Lett. 680 (1996).
When BR in its initial BR570 state is illuminated with visible light having a wavelength near 570 nm, a transient change in the absorbance spectrum occurs, which is referred to as a "photocycle." Photoisomerization of the retinal component occurs from all-trans to 13-cis, with a quantum efficiency of nearly 70%. This leads to a deprotonation of the Schiff base, which causes a substantial shift of the absorbance peak to 410 nm (the M410 state). This is normally the longest-lived state in the photocycle of BR and storage times of several minutes have been reported, but it can be as short as a few milliseconds. See PCT Application No. 93/11470. The BR molecule returns to its initial BR570 state from the M410 state either thermally, or by using blue light to initiate the conversion back to BR570.
The use of BR optical recording materials to record information optically is known in the art. See, for example, U.S. Pat. Nos. 5,470,690, 5,346,789 and 5,374,492 and European Patent No. 487099; see also Downie and Smithey, Ibid.; Downey and Smithey, 35 Appl. Opt. 5780 (1996); and Oesterhelt et al., 24 Q. Rev. Biophysics 425 (1991). Such materials are made by solution-casting, spin-casting, or electro-deposition processes. These materials are used to record information optically, by imaging a pattern into the material using light, and are fully erasable, and so are reusable. The method by which information is recorded is via photochromic processes, i.e., by changes in color of the material upon exposure to light. These materials are useful for holography, digital and analog optical data storage, optical correlators, spatial light modulators, displays, optical switches, optical interconnects, and any other application where the recording or control of light is required.
Optical recording materials have a large number of rigorous performance requirements for use in high-resolution recording, such as optical data storage and holography. A key requirement is the ability to record temporally, physically, and chemically stable spatial patterns that are near the diffraction limit of the recording light, which requires sub-micron resolution. BR materials are promising candidates as high-resolution optical-recording materials. While it is known in the art that BR molecules can record temporally and chemically stable information, bulk materials have not been shown to be physically stable, i.e., the BR molecules migrate over time within a bulk matrix such as a thin film. Such migration destroys optically recorded spatial patterns such as holograms, diffraction gratings and closely-spaced micron-sized bit patterns.
Most of the work on optical device-related applications has relied upon native or genetically engineered BR within the framework of the traditional photocycle, i.e., with use of the purple membrane form. This being the case, recording is achieved primarily by generating the M410 state from the BR570 state. Patterns are first written by photobleaching the BR570 state. The patterns can then be read as positives (with light falling in the spectral region of BR570) or as negatives (by reading with blue light in the spectral region of M410).
Much less is known about another form of BR known as blue membrane. When native BR is either deionized or placed in a low-pH environment (pH&lt;2.6), a dramatically different photocycle and absorbance spectrum result. Blue membrane BR does not form an M410 state or induce a deprotonation of the Schiff base. The initial state of blue membrane, the BR605 state, has an absorbance profile with maximum absorbance at 605 nm.
Genetically engineered BR mutants, such as D85N, D85E, D85Q, R185Q, and many others have similar behavior to that exhibited by low-pH native BR. Some of these BR mutants only exhibit blue membrane forms, i.e., they do not have purple membrane photocycles in any chemical or physical environments.
The photocycle of blue membrane has only two possible states. These states are referred to as the P490 state and the Q390 state, named for the wavelength of their maximum absorbance peaks. The P490 state is photoinduced, while the Q390 state is thermally induced from the P490 state. The rate of conversion from the P490 state to the Q390 state is extremely slow, occurring over several hours at room temperature, but depends on the BR variant that is used. The Q390 state is thermally stable and does not decay in the dark, and so allows relatively permanent recording so long as it is not acted upon by blue light (which causes reversion to the initial BR605 state).
Unlike purple membrane BR materials which have lifetimes that are on the order of hours for the M410 state, blue membrane BR materials have photochromic states (both the P490 and the Q390 states) which are long-lived, exhibiting photochromic lifetimes that are greater than several months. However, just as is the case with purple membrane, the BR molecules within a given matrix tend to diffuse, thereby substantially diminishing the capacity of both types of membrane to permit long-term storage of optical data.
While the prior art discloses a number of methods for the preparation of materials based on BR, none of these methods yield materials which are suitable for long-term storage of optical data. For example, U.S. Pat. No. 5,470,690 describes a process to produce BR materials which have relatively long recording lifetimes using high pH suspensions of polyvinyl alcohol. However, these materials have relatively short photochromic lifetimes, on the order of a few hours. U.S. Pat. No. 5,374,492 describes purple membrane preparations having increased holographic diffraction efficiency by using a genetically engineered mutant which has modifications to amino acid 96 in the BR molecule and uses a proton donor additive having 1-30 wt % water. This preparation has improved diffraction efficiency due to large differences between the initial and the photoinduced absorbance spectra. However, these materials also suffer from limited lifetimes, typically less than a few minutes.
U.S. Pat. No. 5,518,858 describes a photochromic composition comprising an aqueous BR suspension, at least one nitrogen-containing compound, a detergent, and a gelatin binder. This material is claimed to have greater sensitivity to recording light. Again, however, these materials have relatively short lifetimes of less than an hour. PCT Application No. 94/05008 describes fluid compositions of purple membrane BR having increased memory time of more than a day. However, as fluid compositions, they permit relatively fast diffusion of BR molecules through the matrix, thereby thwarting the possibility of long-term storage of optical data.
It is well known that BR molecules can be intramolecularly crosslinked, i.e., whereby the long protein chains are crosslinked to themselves by crosslinking the amino acids within individual molecules in a purple membrane. Packer et al., 145 Biochem. Biophys. Res. Commun. 1164 (1987). These studies indicate that such intracrosslinking of the BR molecule can extend BR's photochromic lifetimes. Sherman et al., 265 Nature 273 (1977).
European Patent Application No. 90 116479.8 describes crosslinking to stabilize an oriented layer of BR for photovoltaic applications such as solar batteries and photosensors, which require protein-oriented BR in order to obtain a photovoltage output from a bulk oriented film. If the protein in the BR film is not oriented, no photovoltage is generated, thus rendering the film useless for its intended purpose. The crosslinking is used to prevent the BR molecules from dissolving in aqueous solutions or from cracking and falling apart after the protein therein has been oriented, which would also render the device useless for its intended purpose.